Biological transformation of anhydrotetracyclines to 5alpha(11alpha)-dehydrotetracyclines



U d S r Pa e BIOLOGICAL TRANSFORMATION OF ANHYDRO- TETRACYCLINES TO a(11a)-DEHYDROTETRA- CY CLINES Philip Andrew Miller and Jerry Robert Daniel McCormick, New City, N.Y., assignors to American Cyanamid Company, New York, N.Y., a corporation of Maine No Drawing. Filed Apr. 6, 1960, Ser. No. 20,269

4 Claims. (Cl. 195-80) This invention relates to a new process for producing 5a(11a)-dehydrotetracyclines and, more particularly is concerned with a novel process for biologically transforming anhydrotetracyclines to 5a(11u)-dehydrotetracyclines as set forth in the following reaction scheme:

011 \JVCONHI I H H\ OH OH O O Anhydrotetracyclines REG 0 plication of Growich and Miller, Serial No. 650,821, filed April 5, 1957, now abandoned. They have been produced by aerobically fermenting an aqueous nutrient medium with certain new mutant strains of S. aureofaciens, the morphological and cultural characteristics of which are adequately described in the aforesaid Growich et a1. application. In addition, viable cultures of these new mutant strains of S. aureofacz'ens, which have been desig nated as S1308, 81308-29, S1308-V146, S1308-V237, and E-504, have been deposited with the American Type Culture Collection in Washington, DC, and have been assigned AT CC accession numbers 12,748, 12,749, 12,750, 12,751 and 13,191 respectively. 7

The 5a(11a)-dehydrotetracyclines possess little or no biological activity. However, since the new mutant strains of S. aureofaciens are high-producing strains frequently producing as much as 9,000l0,000 gammas per milliliter of 7-chloro-5a(l1a)-dehydrotetracycline, considerable efiort has been directed towards converting the 5a(11u)-dehydrotetracyclines into compounds possessing the desired typical broad-spectrum antibacterial activity. One method for accomplishing this result is based upon the discovery that it is possible to catalytically reduce the 5x(11a)-dehydrotetracyclines in good yield with hydrogen in the presence of a noble metal catalyst to the broad spectrum antibiotic tetracycline. A suitable reduction process is described in the copending application of Miller, Serial No. 650,822, filed April 5, 1957. Another method 2,952,587 Patented Sept. 13, 1960 ice for accomplishing this desired result resides in the discovery that it is possible to effect a biological conversion of the 5m(11a)-dehydrotetracyclines under suitable fermentation conditions to the broad-spectrum antibiotics chlortetracycline or tetracycline. A suitable process is described in the copending application of McCormick, Sjolander and Hirsch, Serial No. 650,820, filed' April 5',- 1957. As described in that application, the biological conversion may be effected by the addition of a 5oc(11'a)- dehydrotetracycline compound to a fermentation medium employing a conventional chlortetracycline-synthesizing and tetracycline-synthesizing strain of S. aureofaciens such as the original strain of S. aureofaciens A377 described in United States Patent to Duggar, No. 2,782,055 and deposited at the Northern Regional Research Laboratories, Peoria, Illinois as NRRL 2209. i

The anhydrotetracyclines, the starting materials for the novel process of the present invention, are well known compounds. Anhydrochlorotetracycline is described in United States Patent No. 2,744,931 to Broschard et al., and anhydrotetracycline is described in United States Patent No. 2,744,932 to Waller et al. In addition, the synthesis of dedimethylamino-l2a-deoxy-6-demethylanhydrochlorotetracycline has been-described by J. H. Boothe et al., I.A.C.S. 81, 1006 (1959). The anhydrotetracyclines exhibit antimicrobial activity particularly against certain strains of bacteria which are resistant to the tetracyclines.

Our invention is based upon the discovery that it is'possible, to effect a biological transformation of the anhydrotetracyclines to the 5a(l-luydehydrotetracyclines. This transformation is accomplished by adding an anhydrotetracycline compound to a fermentation medium inoculated with a strain of S. aureofaciens which is capable of producing some one of the 5a(11a)-dehydrotetracyclines, for instance, strain S1308 (ATCC 12748), a producer of 7-Ch1010-5u( 11u)-dehydrotetracycline. After fermenting for a suitable time, for example, from 24 to 96 hours, itis found that the conversion of the anhydrotetracycline'to' the corresponding 5a(11a)-dehydrotetracycline is' about complete.

It is most surprising that the anhydrotetracyclines can serve as substrates which can be acted upon by the'microorganism so as to transform the anhydrotetracycline"compounds to the corresponding 5a(1lbt)-dehydrotetracyclines. In the normal fermentation the ingredients of the nutrient medium serve as the substrate from which the antibiotic is synthesized. It is unexpected to discover that a known stable chemical compound may serve as'the substrate for producing an entirely different tetracycline compound. 7

The conditions of the fermentation for the biological conversion of the anhydrotetracyclines to the 524110;)- dehydrotetracyclines are generally the same as set forth in the aforesaid Growich et al. application and-which, in turn, are generally the same as for the presently known methods of producing chlortetracycline by fermentation. That is, the fermentation medium contains the usual nutrients and mineral substances; Suitable nutrients include starch, dextrose, cane sugar, glucose, molasses, soybean meal, peanut meal, yeast, meat extracts, peptone, urea, cornsteep liquor, distillers solubles, fish meal and other conventional substances. The inorganic salts include such things as calcium carbonate, ammonium sul fate, ammonium chloride, and salts of the various trace elements such as manganese, cobalt, zinc, copper, iron and the like.

The other general conditions of the fermentation, such as hydrogen ion concentration, temperature, time, rate of aeration, preparation of the inoculum, sterilization, inoculation and the like are conventional and are simi- 3 lar to those for the production of chlortetracycline shown in the U.S. Patent No. 2,482,055 to Duggar.

After the fermentation has been continued for the desired period of time'and the transformation of the anhydrotetracycline compound to the corresponding oz(11oc)- dehydrotetracycline is substantially complete, the 50t(110c)- dehydrotetracycline compound may be isolated from the fermentation mash in any convenient manner. A suitable recovery process is described in the aforesaid Growich et al; application which, briefly, consists in acidifying the fermentation liquor by adjusting the pH to from 1 to 2 with a mineral acid, filtering the mash, and extracting the aqueous filtrate with an alcohol, such as n-butanol. The extract is then chromatographed on a column in the usual manner and the column is developed with a mixture of 80% n-butanol and 20% chloroform,

The eluted fractions are concentrated and freeze-dried. The freeze-dried material is dissolved in methanol and crystallization of the 5a(11a)-dehydrotetracycline compound as the free base takes place rapidly. The crystals are filtered, washed and dried, and may be recrystallized I from alcohol in a standard manner.

The invention will be described in greater detail in conjunction with the following specific examples;

' A 7 Example 1 V Spores of a strain of S. aureofaciens (S1308, ATCC No. 12,748) are used to inoculate 100 milliliters of a sterile medium prepared according to the following formulation: a

Ingredients; 7 Amounts, grams Sucrose 30 V Ammonium sulfate 2 Calcium carbonate 7 Corn steep liquor 20 Tap water q.s., 1000 milliliters. After incubation at 265 C. for'24 hours, 1 milliliter portions of this inoculum are used to seed 25 milliliters of a sterile medium prepared according to the following formulation: 7 I 7 Ingredients: Amounts, grams Ammonium sulfate 5 Calcium carbonate 9 Ammonium chloride 1.5 Magnesium chloride.6I-I O 2 Ferrous sulfate.7H O 12 Manganese sulfate.4H O

Cobalt chloride.6H O 1 Zinc sulfate.7H O 20 Corn steep liquor 25 -30 Starch 55 Water q.s., 1000 milliliters.

Three milliliter portions of the mash are transferred 4 viously buffered by dipping in 0.3 M NaH PO solution adjusted to pH 3.0 with phosphoric acid, and then air dried. The strip is then developed for 18 hours with n-butanol which is saturated with the phosphate solution described above. The air-dried chromatogram is scanned for radioactivity and shows the disappearance of C anhydrochlortetrac ycline (R; 0.70) and the concomitant appearance of C -7-chloro-5a(11a)-dehydrotetracycline (R; 0.44).

Example 2 The procedure or, the preceding example is repeated using a vegetative inoculum of S. aureofaciens (strain S1308, ATCC No. 12,748) except that 1.5 milligrams of C -labeled anhydrotetracycline (R; 0.60) is used instead of the anhydrochlortetracycline used in Example 1. The fermentation is carried out as described in Example 1 and paper strip chromatography shows the presence of C -5a(11a)-dehydrotetracycline (R, 0.37).

Example 3 The procedure of Example 1 is repeated using a vegetative inoculum of S. aureofaciens (strain E-504, ATCC No. 13,191). In addition, 1.5 milligrams ofBr -labeled anhydrobromtetracycline (R; 0.70) is used instead of the anhydrochlorteu'acycline used in Example 1. The fermentation is carried out as described in Example 1 and paper strip chromatography shows the presence of Br -7-bromo-5a(11a) dehydrotetracycline (R, 0.44).

to 20 x150 mm. sterile shaker tubes containing 1.5 milligrams of C -labeled anhydrochlortetracycline. The tubes are mounted on a reciprocating shaker and allowed to incubate for an additional 72 hours at 25 C. The, tubes are then removed, the mash in each diluted with 10 milliliters of 0.1 N hydrochloric acid, and the myceliurn removed by filtrationg The filtrate is extracted by shaking with 0.5 milliliterof n-chlorophenol. Five microlitersof thephenol phase are spotted on a sheet of Whatman No. 1 chromatographic paperwhich is pre- We claim:

1. The process for the biological transformation of an anhydrotetracycline to the corresponding 5a(11a)-dehydrotetracycline which comprises adding an anhydrotetracycline to an aqueous nutrient medium, aerobically fermenting the aqueous nutrient medium with a 5a(11u)-dehydrotetracycline-producing strain of S. aureofaciens, and continuing the fermentation until the anhydrotetracycline is substantially converted to the corresponding 5oc(l1oz)- dehydrotetracycline.

2. The process for the biological transformation of anhydrotetracycline to 5a(11a)-dehydrotetracycline which comprises adding crystalline anhydrotetracycline to an aqueous nutrient medium, aerobically fermenting the aqueous nutrient medium with a 5a(11a)-dehydrotetracycline-producing strain of S. aureofaciens, and continuing the fermentation until the anhydrotetracycline is substantially converted to 5oc( 11a)-dehydrotetracycline.

3. The process for the biological transformation of 'anhydrochlortetracycline to 7-chloro 5041111) dehydrotetracycline which comprises addin crystalline anhydrochlortetracycline to an aqueous nutrient medium, aerobically fermenting the aqueous nutrient medium with a 7-chloro-5a(l1u) -dehydrotetracycline-producing strain of S. aureofaciens, and continuing the fermentation until the anhydrochlortetracycline is substantially converted to 7- chloro-5a(llaydehydrotetracycline.

4. The process for the biological transformation of anhydrobromtetracycline to 7-bromo-5 rx( 1 10a) -dehydrotetracycline which comprises adding crystalline anhydrobromtetracycline to an aqueous nutrient medium, aerobically fermenting the aqueous nutrient medium with a 7-bromo- 5oz(11oc) -dehydrotetracycline-producing strain of S. aureofaciens, and continuing the fermentation until the anhydrobromtetracycline is substantially converted to 7-bromo-S a 1 1 a) dehydrotetracycline.

No references cited.

IERNEST w; sw pER DAVID L. LADD UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 2352 587 September 13 1960 Philip Andrew Miller et al0 It is hereby certified that error appears in the printed specification of the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

In the heading to the printed specification line in the title of invention for "50;(llc read 5a(lla)=- column l, lines 17 19 39., 42 and 57 for v"51(ll 1)=-" each occurrence read 5a(lla) same column I lines 61 63 and 67 for "5(I(llU,)-'" each occurrence, read 5a(lla)=- column 2 lines 3 and 9 for "5 1(llc1)==" each occurrence read 5a(lla)- line 23 for "-=-l2q-deoxy=-" read -l2a=- deoxylines .31 and 37 for "SqdllqO- each occurr= ence read 5a(lla) same column 2 lines 4O and 53 for "5d(lld)-= each occurrence read 5a(lla)=-= column 3 lines 5 6 and 19 for "5 1(lla)=' each occurrence, read 5-a(lla) line 65 for "n -chlorophenol" read p== chlorophenol column 4, lines 8 18, 28 31 34 and 37 for "5(I(llCI)-" each occurrence read 5a(lla)- same column 4 lines 40 43 46 and 48 for "5(I(lld each occurrence read 5a(lla)-- lines 52 55 and 57 for '5(1(1101)" each occurrence read 5a(1la)- lines 61 and 64 for "5 1(l1a)", each occurrence read 5a(lla)-= Signed and sealed this 181311 day of April 1961.,

(SEAL) Attest:

Attesting Oflicer Commissioner of Patents 

1. THE PROCESS FOR THE BIOLOGICAL TRANSFORMATION OF AN ANHYDROTETRACYCLINE TO THE CORRESPONDING 5A(11A)-DEHYDROTETRACYCLINE WHICH COMPRISES ADDING AN ANHYDROTETRACYCLINE TO AN AQUEOUS NUTRIETNT MEDIUM, AEROBICALLY FERMENTING THE AQUEOUS NUTRIENT MEDIUM WITH A 5A(11A)-DEHYDROTETRACYCLINE-PRODUCING STRAIN OF S. AUREOFACIENS, AND CONTINUING THE FERMENTATION UNTIL THE ANHYDROTETRACYCLINE IS SUBSTANTIALLY CONVERTED TO THE CORRESPONDING 5A(11A)DEHYDROTETRACYCLINE. 